Balamuthia mandrillaris: In Vitro Interactions with Selected Protozoa and Algae

<i>Balamuthia mandrillaris</i>exhibits metalloprotease activities

The free-living amoeba Balamuthia mandrillaris is a rare but highly lethal agent of amoebic encephalitis in humans and many other mammalian species. Here, we announce the first draft genome sequence of the original 1990 isolate cultured from the brain of a deceased mandrill baboon.

Balamuthia mandrillaris is a free-living ameba that causes encephalitis in humans (both immunocompetent and immunocompromised), horses, dogs, sheep, and nonhuman primates. The ameba is present in soil and likely is transmitted by inhalation of airborne cysts or by direct contamination of a skin lesion. Approximately 150 cases of balamuthiasis have been reported worldwide since recognition of the disease in 1990. Balamuthiasis is difficult to diagnose because 1) the clinical symptoms mimic those of several other types of encephalitis, 2) few laboratories perform appropriate diagnostic testing, and 3) many physicians are unaware of the disease. The lack of recognition and subsequent delay in diagnosis might be a factor in its high mortality. Since 1998, the California Encephalitis Project (CEP) has been testing encephalitis cases for both common and uncommon agents known to cause encephalitis, including Balamuthia. This report describes the 10 balamuthiasis cases identified by CEP during 1999-2007. The preliminary diagnoses in these cases included neurotuberculosis, viral meningoencephalitis, neurocysticercosis, and acute disseminated encephalomyelitis. All but one patient died. These findings underscore the importance of increasing awareness among clinicians, epidemiologists, and public health officials for timely recognition and potential treatment of Balamuthia encephalitis.

Peroxisomes perform various metabolic processes that are primarily related to the elimination of reactive oxygen species and oxidative lipid metabolism. These organelles are present in all major eukaryotic lineages, nevertheless, information regarding the presence of peroxisomes in opportunistic parasitic protozoa is scarce and in many cases it is still unknown whether these organisms have peroxisomes at all. Here, we performed ultrastructural, cytochemical, and bioinformatic studies to investigate the presence of peroxisomes in three genera of free-living amoebae from two different taxonomic groups that are known to cause fatal infections in humans. By transmission electron microscopy, round structures with a granular content limited by a single membrane were observed in Acanthamoeba castellanii, Acanthamoeba griffini, Acanthamoeba polyphaga, Acanthamoeba royreba, Balamuthia mandrillaris (Amoebozoa), and Naegleria fowleri (Heterolobosea). Further confirmation for the presence of peroxisomes was obtained by treating trophozoites in situ with diaminobenzidine and hydrogen peroxide, which showed positive reaction products for the presence of catalase. We then performed comparative genomic analyses to identify predicted peroxin homologues in these organisms. Our results demonstrate that a complete set of peroxins-which are essential for peroxisome biogenesis, proliferation, and protein import-are present in all of these amoebae. Likewise, our in silico analyses allowed us to identify a complete set of peroxins in Naegleria lovaniensis and three novel peroxin homologues in Naegleria gruberi. Thus, our results indicate that peroxisomes are present in these three genera of free-living amoebae and that they have a similar peroxin complement despite belonging to different evolutionary lineages.

Heme is an essential molecule for most organisms, yet some parasites, like Trypanosoma cruzi, the causative agent of Chagas disease, cannot synthesize it. These parasites must acquire heme from their hosts, making this process critical for their survival. In the midgut of the insect vector, T. cruzi epimastigotes are exposed to both hemoglobin (Hb) and free heme resulting from its degradation. Despite the importance of this nutrient, how different heme sources influence parasite gene expression remains poorly understood.Here, we showed that heme restitution either as hemin or Hb to heme-starved parasites induces an early and distinct transcriptional response in T. cruzi epimastigotes. Using RNA sequencing at 4- and 24-hours post-supplementation, we identified gene subsets commonly or uniquely regulated by each heme source, including genes putatively linked to heme acquisition and metabolism. The study includes the first focused characterization of CRAL/TRIO domain-containing protein (TcCRAL/TRIO), a novel heme-responsive hemoprotein. Our results provide a more detailed picture of T. cruzi biology and highlights heme acquisition as a promising point of vulnerability to control parasite proliferation.

Unstimulated mouse peritoneal exudate cells were cultured on coverslips in Medium 199 containing 10 per cent (v/v) calf serum. Cytochalasin B dissolved in dimethyl sulphoxide (DMSO) and diluted in Medium 199 was added to cultures to give final concentrations of 1, 5 and 10 mug/ml. Equal numbers of Leishmania mexicana promastigotes, Trypanosoma cruzi epimastigotes and sheep red cells were added to 24 hr cultures incubated at 37 C. The macrophage monolayers were fixed and stained at various time intervals. L. mexicana promastigotes sheep red blood cells were found to attach to macrophages in the presence of the drug but did not enter the cells. When the medium containing the cytochalasin was replaced with normal medium phagocytosis of the adherent parasites and red cells followed rapidly. T. cruzi epimastigotes were found inside macrophages in both drug-treated and drug-free cultures although the number found to be intracellular in the latter was significantly greater. This study suggest that L. mexicana promastigotes enter macrophages by being phagocytosed, whereas T. cruzi epimastigotes can actively penetrate these cells.

Both Eimeria tenella and Toxoplasma gondii are common apicomplexan parasites in chickens. Host cell invasion by both protozoans includes gliding motility, host cell attachment and active penetration. Chicken macrophages as phagocytic cells participate in the innate host immune response against these two parasites. In this study, primary chicken monocyte-derived macrophages (MM) were infected with both pathogens to investigate mutual and host-parasite interactions. MM cultures were assigned to groups that were infected with E. tenella, T. gondii or both. In co-infected cultures, MM were first exposed to E. tenella sporozoites for 2 h. Afterwards, T. gondii tachyzoite infection was performed. Live-cell imaging was carried out to observe cell invasion and survival of T. gondii by single parasite tracking over a period of 20 h post infection (hpi). Quantitative analysis for parasite replication was performed by real-time quantitative PCR (qPCR) at 2, 6, 12 and 24 hpi. Overall, the ability of T. gondii to penetrate the cell membrane of the potential host cell was reduced, although high motility was displayed. We found that T. gondii tachyzoites adhered for more than 4 h to macrophages during early co-infection. qPCR results confirmed that significantly less T. gondii entered in E. tenella-activated MM at 2 hpi, and a reduced proportion of intracellular T. gondii survived and replicated in these cells at 24 hpi. We conclude that E. tenella modulates host cell responses to another apicomplexan agent, T. gondii, reducing active invasion and multiplication in chicken primary macrophages.

Balamuthia mandrillaris is a free-living ameba and an opportunistic agent of lethal granulomatous amebic encephalitis (GAE) in humans and other mammals. Its supposed routes of infection have been largely assumed from what is known about Acanthamoeba spp. and Naegleria fowleri, other free-living amebae and opportunistic encephalitis agents. However, formal proof for any migratory pathway, from GAE patients or from animal models, has been lacking. Here, immunodeficient mice were infected with B. mandrillaris amebae by intranasal instillation, the most likely natural portal of entry. By means of classical and immunohistology, the amebae are shown to adhere to the nasal epithelium, progress along the olfactory nerves, traverse the cribriform plate of the ethmoid bone, and finally infect the brain. A similar invasion pathway has been described for N. fowleri. The data suggest that the olfactory nerve pathway is a likely route for natural infection of the brain by B. mandrillaris amebae.

Influence of monosaccharides on the infection of vertebrate cells by Trypanosoma cruzi and Toxoplasma gondii

BackgroundReservosomes are lysosome-related organelles found in Trypanosoma cruzi epimastigotes. They represent the last step in epimastigote endocytic route, accumulating a set of proteins and enzymes related to protein digestion and lipid metabolism. The reservosome matrix contains planar membranes, vesicles and lipid inclusions. Some of the latter may assume rectangular or sword-shaped crystalloid forms surrounded by a phospholipid monolayer, resembling the cholesterol crystals in foam cells.Methodology/Principal FindingsUsing Nile Red fluorimetry and fluorescence microscopy, as well as electron microscopy, we have established a direct correlation between serum concentration in culture medium and the presence of crystalloid lipid inclusions. Starting from a reservosome purified fraction, we have developed a fractionation protocol to isolate lipid inclusions. Gas-chromatography mass-spectrometry (GC-MS) analysis revealed that lipid inclusions are composed mainly by cholesterol and cholesterol esters. Moreover, when the parasites with crystalloid lipid-loaded reservosomes were maintained in serum free medium for 48 hours the inclusions disappeared almost completely, including the sword shaped ones.Conclusions/SignificanceTaken together, our results suggest that epimastigote forms of T. cruzi store high amounts of neutral lipids from extracellular medium, mostly cholesterol or cholesterol esters inside reservosomes. Interestingly, the parasites are able to disassemble the reservosome cholesterol crystalloid inclusions when submitted to serum starvation.

To investigate the possible role of hepatocyte growth factor (HGF)in the reconstruction process following inflammatory damage in lung tissue, we compared HGF production of human lung microvascular endothelial cells (HL MECs) and human umbilical vein endothelial cells (HUVECs) after stimulation by interleukin(IL)-1 β. In an HL MEC-conditioned medium, largeamounts of total (single and 2-chain) HGF were detected, and were 26- to 28-fold higher than those in HUVECs or human lung fibroblasts. The production of total HGF increased in a dose-dependent manner (4.7 to 9.2 times) with IL-1 β. In contrast, the amount of HGF in an HUVEC-conditioned medium was unaffected by IL-1 β treatment. The amount of cell-associated HGF also showed a dose-related increase (140% to 160%) in HL MECs, but not in HUVECs with IL-1 β. In addition, HGF and c-met (HGF receptor) mRNAs in HL MECs and HUVECs were examined by the RT-PCR method. HGF and c-met mRNAs were clearly detected in HL MECs before and after treatment with IL-1 β, but not in HUVECs. These results suggest that increases in HGF production from HL MECs may play a role in the reconstruction process following inflammatory damage in lung tissue.

Three enzyme immunoassays (EIAs) for diagnosis of Chagas' disease were developed with fixed forms of Trypanosoma cruzi using a panel of 435 sera from the following groups: Venezuelan subjects positive by immunofluorescence (n = 70), Venezuelan healthy controls (n = 85), healthy Canadians (n = 166), and subjects with other parasitic diseases (n = 114). All assays achieved 100% sensitivity and reasonable specificity for amastigotes (97.6%), epimastigotes (98.3%), and trypomastigotes (99.3%). The fixed-trypomastigote assay was stable over 4 months at 4 degrees C and room temperature. These data suggest that a fixed-trypomastigote EIA may be a suitable candidate for blood bank screening.

Chagas disease affects about 7 million people worldwide. Only two drugs are currently available for the treatment for this parasite disease, namely, benznidazol (Bzn) and nifurtimox (Nfx). Both drugs have limited curative power in the chronic phase of the disease. Therefore, continuous research is an urgent need so as to discover novel therapeutic alternatives. The development of safer and more efficient therapeutic anti-T. cruzi drugs continues to be a major goal in trypanocidal chemotherapy. Synthesis, 2D-QSAR and drug-like physicochemical properties of a set of quinazolinone and quinazoline derivatives were studied as trypanocidal agents. All compounds were screened in vitro against Trypanosoma cruzi (Tulahuen strain, Tul 2 stock) epimastigotes and bloodstream trypomastigotes. Out of 34 compounds synthesized and tested, six compounds (5a, 5b, 9b, 9h, 13f and 13p) displayed significant activity against both epimastigotes and tripomastigotes, without exerting toxicity on Vero cells. The antiprotozoal activity of these quinazolinone and quinazoline derivatives represents an interesting starting point for a medicinal chemistry program aiming at the development of novel chemotherapies for Chagas disease.

Our laboratories have been investigating biological effects of a sesquiterpenoid polygodial and its natural and synthetic analogues. Herein, we report the evaluation of these compounds against the three forms of Trypanosoma cruzi, amastigotes, trypomastigotes and epimastigotes. Although polygodial was found to be poorly active, its natural congener epipolygodial and synthetic Wittig-derived analogues showed low micromolar potency against all three forms of the parasite. Synthetic α,β-unsaturated phosphonate 9 compared favorably with clinically approved drugs benznidazole and nifurtimox, and was effective against trypomastigotes, toward which benznidazole showed no activity.

Improving human coronavirus OC43 (HCoV-OC43) research comparability in studies using HCoV-OC43 as a surrogate for SARS-CoV-2